Phosphorylation of PPARg in epididymal white adipose tissue in ob/ob mice after treatment with MEK inhibitors. Gene expression in ob/ob epididymal white adipose tissue after treatment with vehicle or either of two MEK inhibitors, PD0325901 or GSK1120212 (n = 7, 7 and 8, respectively). Areas under the curve and gene expression were analysed by analysis of variance.

Nature 2015 517(7534), 391-5. PD0325901 purchased from Selleck

SMYD3 knockout augments the effects of the MEK1/2 inhibitor Trametinib (GSK1120212) in vivo. Representative serial HE staining and IHC for pERK1/2, a marker of Ras activity, and MUC5, a marker of PanIN lesions. All scale bars, 50 um.

Activity of vorinostat in Jurkat and Peer T-ALL cell lines in an MTT cell viability assay. Cells were treated with increasing drug concentrations for 72 h. The data are plotted as the mean % of DMSO-treated control cells against the corresponding drug concentration. The error bars are the standard error. For each drug and cell line, 3-4 independent experiments were performed with 6 replicates at each drug concentration.

SAHA and other nonselective HDAC inhibitors increased steady-state acetylation of tubulin and histones manifested by the staining of acetylated microtubules and punctuate nuclear staining of acetylated histone.

Although vemurafenib treatment decreased the volume of sensitive tumours (A375 alone)(b), Green fluorescent protein (GFP) staining confirmed increased numbers of resistant cells in regressing tumours, and EdU or BrdU staining confirmed their increased proliferation rate compared to the vehicletreated controls (c). Tumours comprising only resistant cells showed no growth difference when treated with vehicle or vemurafenib (d), indicating that the growth advantage of resistant cells in regressing tumours was not caused by direct effects of vemurafenib on cancer or stromal cells. In line with these findings, A375R cells co-implanted with other vemurafenib-sensitive melanoma cell lines (Colo800, LOX and UACC62) also showed an up toeightfold growthincreasecompared to vehicle-treated control groups (e). Local growth acceleration of resistant cells in the regressing subcutaneous tumours resulted in higher lung metastatic burden (f).

This analysis highlighted FRA1 (also known FOSL1), a member of the AP1 transcription factor complex and effector of the ERK pathway27, as one of the putative upstream regulators of the TIS .FRA1 was downregulated in all drug-sensitive cells, but not in resistan cells, treated with vemurafenib, crizotinib and erlotinib (c, d).

Phosphorylation of PPARg in epididymal white adipose tissue in ob/ob mice after treatment with MEK inhibitors. Gene expression in ob/ob epididymal white adipose tissue after treatment with vehicle or either of two MEK inhibitors, PD0325901 or GSK1120212 (n = 7, 7 and 8, respectively). Areas under the curve and gene expression were analysed by analysis of variance.

Rating

Source

Nature, 2015, 517(7534), 391-5. PD0325901 purchased from Selleck

Method

Western blot

Cell Lines

Metabolic

Concentrations

10 mg/kg

Incubation Time

5 days

Results

PD0325901 caused a decrease in PPARc phosphorylation at S112 and S273, confirming the established role of ERKs in regulating S112 and strongly suggesting a new role in regulating S273 (refs 22-24).

SMYD3 knockout augments the effects of the MEK1/2 inhibitor Trametinib (GSK1120212) in vivo. Representative serial HE staining and IHC for pERK1/2, a marker of Ras activity, and MUC5, a marker of PanIN lesions. All scale bars, 50 um.

Administration of Kras and Kras;Smyd3 mutant mice with a normal dose of Trametinib blocked tumorigenesis in both strains, though phosphorylation of ERK1/2 was still lower in mice depleted of SMYD3. Notably, a low dose Trametinib regimen, which only partially inhibited pERK1/2 levels and the formation of neoplastic lesions in Kras mutant mice, was sufficient to block tumorigenesis and ERK1/2 activation in Smyd3 knockouts.

Activity of vorinostat in Jurkat and Peer T-ALL cell lines in an MTT cell viability assay. Cells were treated with increasing drug concentrations for 72 h. The data are plotted as the mean % of DMSO-treated control cells against the corresponding drug concentration. The error bars are the standard error. For each drug and cell line, 3-4 independent experiments were performed with 6 replicates at each drug concentration.

It examined responsiveness to dexamethasone and the class I/II HDAC inhibitor vorinostat in a panel of T-ALL cell lines with wild type or mutant CREBBP alleles. This demonstrated sensitivity to vorinostat at clinically useful concentrations (IC50 below 1礛) in the majority of cell lines tested.

Ectopic COT expression in A375 and SKMEL28 cells also conferred decreased sensitivity to the MEK inhibitors CI-1040 and AZD6244, suggesting that COT expression alone was sufficient to induce this phenotype. In the setting of ectopic COT expression, exposure to AZD6244 or CI-1040 in combination with PLX470 (1 μM each) reduced cell growth and pERK expression more effectively than did single-agent PLX4720, even at concentrations of 10 μM.